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Expression Stability of Reference Genes for Quantitative RT-PCR of Healthy and Diseased Pituitary Tissue Samples Varies Between Humans, Mice, and Dogs

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Abstract

Pituitary surgery generates pituitary tissue for histology, immunohistochemistry, and molecular biological research. In the last decade, the pathogenesis of pituitary adenomas has been extensively studied in humans, and to a lesser degree in dogs, and tumor oncogenesis has been studied in knock-out mice, often by means of quantitative reversed-transcriptase PCR (RT-qPCR). A precondition of such analyses is that so-called reference genes are stably expressed regardless of changes in disease status or treatment. In this study, the expression of six frequently used reference genes, namely, tata box binding protein (tbp), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (ywhaz), hydroxymethylbilane synthase (hmbs), beta-2-microglobulin (b2m), succinate dehydrogenase complex subunit A (sdha), and glyceraldehyde 3 phosphate dehydrogenase 1 (gapdh), was studied in pituitary tissue (normal and adenoma) from three species (humans, mice, and dogs). The stability of expression of these reference genes differed between species and between healthy and diseased tissue within one species. Quantitative analysis based on a single reference gene that is assumed to be stably expressed might lead to wrong conclusions. This cross-species analysis clearly emphasizes the need to evaluate the expression stability of reference genes as a standard and integral aspect of study design and data analysis, in order to improve the validity of the conclusions drawn on the basis of quantitative molecular analyses.

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Acknowledgments

We thank Dr. A. de Bruin, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands, for generously donating the mice samples. We thank Jane Sykes for proofreading of the manuscript.

Disclosures

None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

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Authors and Affiliations

  1. Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD, Utrecht, The Netherlands

    Sarah J. van Rijn, Frank M. Riemers, Douwe van den Heuvel, Jeannette Wolfswinkel, Björn P. Meij & Louis C. Penning

  2. Department of Internal Medicine, Division of Endocrinology, Erasmus Medical Center, PO Box 2040, 3000 CA, Rotterdam, The Netherlands

    Leo Hofland

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  1. Sarah J. van Rijn
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  2. Frank M. Riemers
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  3. Douwe van den Heuvel
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  4. Jeannette Wolfswinkel
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  6. Björn P. Meij
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Correspondence to Sarah J. van Rijn.

Additional information

Björn P. Meij and Louis C. Penning equally contributed to this work.

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van Rijn, S.J., Riemers, F.M., van den Heuvel, D. et al. Expression Stability of Reference Genes for Quantitative RT-PCR of Healthy and Diseased Pituitary Tissue Samples Varies Between Humans, Mice, and Dogs. Mol Neurobiol 49, 893–899 (2014). https://doi.org/10.1007/s12035-013-8567-7

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  • DOI: https://doi.org/10.1007/s12035-013-8567-7

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